Methods and compositions for inhibiting CD14 mediated cell activation

ABSTRACT

This invention provides hybridoma cell lines producing monoclonal antibodies which inhibit CD14 mediated cell activation. Monoclonal antibodies produced by these cell lines also are provided. The antibodies are useful for the detection of the presence of cell surface and soluble CD14 in a sample. Chimeric and CDR grafted antibodies generated from the above monoclonal antibodies are further provided. Pharmaceutical compositions containing the above biological compositions are provided. These are useful to treat and prevent disorders with CD14 mediated cell activation, such as sepsis.

This application is a continuation of application Ser. No. 08/070,160,filed on May 28, 1993.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods and compositions for inhibitingCD14 mediated cell activation. More particularly, the present inventionrelates to molecules that bind the CD14 monocyte antigen at a site whichinhibits CD14 mediated cell activation.

2. Description of the Related Art

The correct functioning of a cell depends partly on its ability tocommunicate with its environment; external stimuli often interact withmembrane receptors which, in turn, induce second messengers thatultimately affect transcription factors. The latter then activate orrepress the expression of certain genes resulting in a specific patternof proteins in a given cell.

The transcription factor NF-κB (nuclear factor-κB) is induced by avariety of stimuli to contact its DNA-binding motif and regulate a setof genes encoding immunoreceptors, cytokines, and viral proteins.Included among the various factors which can activate NF-κB islipopolysaccharide (LPS). LPS, in turn, is intimately involved in theinduction of the sepsis syndrome, including septic shock, systemicinflammatory response syndrome, and multiorgan failure.

Sepsis is a morbid condition induced by a toxin, the introduction oraccumulation of which is most commonly caused by infection or trauma.The initial symptoms of sepsis typically include chills, profuse sweat,irregularly remittent fever, prostration and the like, followed bypersistent fever, hypotension leading to shock, neutropenia, leukopenia,disseminated intravascular coagulation, acute respiratory distresssyndrome and multiple organ failure.

Lipopolysaccharide, or endotoxin, is a toxic component found in theouter membrane of all gram-negative microorganisms (e.g., Escherichiacoli Klebsiella pneumonia, Pseudomonas aeruginosa). It has beendetermined that LPS is a potent and pleiotropic stimulus for immunecells, both in vitro and in vivo (Morrison, D. C. & J. L. Ryan, Annu.Rev. Med., 38:417, 1987, Bone, R. C., Ann. Intern. Med., 115:457, 1991).Compelling evidence supports the toxic role of LPS in that all of thepathophysiological effects noted in humans during gram-negative sepsiscan be completely duplicated with purified LPS. The mechanism by whichthis toxic component activates responsive cells is complex and not fullyunderstood. The host response to gram-negative bacterial infection isdependent upon effector cell recognition of these bacteria and/or LPSand involves serum proteins and cell membrane receptors. While theclearance of bacteria and LPS is via endocytosis and phagocytosis byreticuloendothial cells, concomitant activation of the host immuneresponse by LPS results in secretion of cytokines by activatedmacrophages which can trigger the exaggerated host responses that occurduring gram-negative bacterial infection.

The discovery by Tobias, et al. (J. Exp. Med., 164:777, 1986) of a serumprotein, identified as LPS binding protein (LBP), that exhibits highaffinity binding to LPS (K_(d)=10⁻⁹ M⁻¹), helped to define the fate ofLPS once released in vivo. It was demonstrated that this novel protein,with a molecular weight of 60 kD, which is synthesized in the liver isan acute phase serum protein reaching levels of 200 μg/ml in humans. Theformation of high affinity LPS/LBP complexes is followed by recognitionby macrophages with subsequent release of TNF-α and other macrophagesecretory products (Schumann, R. R., et al., Science, 249:1429, 1990).Additional studies on the effects of LPS complexed with LPB led to thediscovery of its specific receptor on the surface of monocytes andmacrophages; CD14 (Wright, S. D., et al., Science, 249:1431, 1990).Further analysis with mAbs specific for CD14 revealed that the domain towhich one anti-CD14 mAb (3C10; VanVoorhis, W. C., et al., J. Exp. Med.,158:126, 1983) bound was part of, or in close proximity to, the LPS/LBPbinding site on CD14. Monoclonal antibody 3C10, by nature of its abilityto block LPS/LBP binding to CD14, was capable of inhibiting TNF-αrelease in a human whole blood assay, after stimulation with LPS. It issuggested by this discovery that the blocking of a single proteindeterminant (the ligand binding site on CD1 4) is sufficient, even inthe presence of all other cells, proteins and factors contained in humanwhole blood, to inhibit TNF-α release (known to be a key mediator inseptic shock) and other macrophage secretory products in response toLPS.

In spite of the advances which have been made in understanding thenature of CD14 mediated cell activation disorders, such as sepsis,considerable need remains for compositions which can be used to inhibitsuch activation and to diagnose these disorders. The present inventionprovides such compositions.

SUMMARY OF THE INVENTION

This invention provides hybridoma cell lines producing monoclonalantibodies, the monoclonal antibodies being capable of inhibiting CD14mediated cell activation. Monoclonal antibodies produced by these celllines also are provided. These monoclonal antibodies are broadly usefulin inhibiting NF-κB activation by a ligand which binds to CD14 and wouldotherwise be capable of inducing NF-κB activation. Biologically activefragments of the monoclonal antibodies are provided. The antibodies andfragments are useful for the detection of the presence of cellsurface-associated and soluble CD14 in a sample. Chimeric and CDRgrafted antibodies generated from the above monoclonal antibodies arefurther provided.

Pharmaceutical compositions containing the above biological compositionsare provided. These are useful to treat and prevent LPS-associateddisorders, such as sepsis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is the nucleic acid sequence for the human soluble CD14 receptor(SEQ ID NO:9).

FIG. 2 is the nucleic acid and amino acid sequence of the 28C5 heavychain (SEQ ID NOS:1 and 2, respectively).

FIG. 3 is the nucleic acid and amino acid sequence of the 28C5 lightchain (SEQ ID NOS:3 and 4, respectively).

FIG. 4 is the nucleic acid and amino acid sequence of the 18E12 heavychain (SEQ ID NOS:5 and 6, respectively).

FIG. 5 is the nucleic acid and amino acid sequence of the 18E12 lightchain (SEQ ID NOS:7 and 8, respectively).

FIG. 6 is FACS analysis of control THP-1 cells only.

FIG. 7 is FACS analysis of control THP-1 cells and FiTC conjugate only.

FIG. 8 is FACS analysis of positive control THP-1 and MY4 antibody.

FIG. 9 is FACS analysis of 28C5 antibody.

FIG. 10 is FACS analysis of 18E12 antibody.

FIG. 11 shows the titers of various mAb to sCD14.

FIG. 12 shows the results of a competition assay between mAb 3C10 and apanel of anti-CD14 mAbs.

FIG. 13 shows the blocking of LPS/LBP binding to CD14 by anti-CD14 mAbs.

FIG. 14 shows the results of an evaluation of the ability of anti-CD14mAbs to block cytokine release in HL-60 cells.

FIG. 15 shows effect of anti-CD14 mAbs to inhibit LPS binding tocellular CD14.

FIG. 16 shows effect of anti-CD14 mAbs on LPS-dependent, CD14-mediatedactivation of cells.

FIG. 17 shows the mean arterial pressure of monkeys challenged with LPSand treated with 18E12 (●), 28C5 (▪) or IgG1 (x).

FIG. 18 shows pre and post human IFN-γ treatment CD14 levels and LBPlevels in a monkey.

FIG. 19 shows the lavage/plasma ration of BSA in monkeys treated with18E12, 28C5 or IgG1.

FIG. 20 shows the antibody half life of 18E12, 28C5 and IgG1 in monkeys.

FIG. 21 shows CD14 levels in monkeys treated with antibody (18E12, 28C5and IgG1) alone (top) or challenged with LPS after antibody treatment(bottom).

FIG. 22 shows LBP levels in monkeys treated with antibody (18E12, 28C5and IgG1) alone (top) or challenged with LPS after antibody treatment(bottom).

FIG. 23 shows ALT/GPT levels in monkeys treated with antibody (18E12,28C5 and IgG1) alone (top) or challenged with LPS after antibodytreatment (bottom).

FIG. 24 shows E-selectin levels in monkeys treated with antibody (18E12,28C5 and IgG1) alone (top) or challenged with LPS after antibodytreatment (bottom).

FIG. 25 shows TNF levels in monkeys treated with antibody (18E12, 28C5and IgG1) and challenged with LPS (top) and IL-1 levels in monkeystreated with antibody (18E12, 28C5 and IgG1) and challenged with LPSafter antibody treatment (bottom).

FIG. 26 shows IL-6 levels in monkeys treated with antibody (18E12, 28C5and IgG1) and challenged with LPS (top) and IL-8 levels in monkeystreated with antibody (18E12, 28C5 and IgG1) and challenged with LPSafter antibody treatment (bottom).

FIG. 27 shows inhibition of TNF release in human whole blood stimulatedwith LPS by treatment with 18E12 (●), 28C5 (▴) or 23G4 (▪).

FIG. 28 shows the effect of anti-CD14 antibodies on LPS-induced TNFsecretion in baboon blood. (23G4 (●), 28C5 (∇), and 18E12 (▾).

FIG. 29 shows the amino acid sequence of the light chains of monoclonalantibodies 3C10, 28C5, 23G4 and 18E12.

FIG. 30 shows the amino acid sequence of the heavy chains of monoclonalantibodies 3C10, 28C5, and 18E12.

DETAILED DESCRIPTION OF THE INVENTION

A full length polypeptide for a human soluble CD14 (“sCD14”) is providedby the disclosure. As used herein, “CD14” means the cell surfacereceptor that has been identified as the binding site for LPS when theLPS is present as an LPS:LBP complex. The CD14 cell surface receptor isa glycerophosphatidylinositol (GPI)-linked protein present on thesurface of mature monocytes, neutrophils, and macrophages. Native CD14also is spontaneously released from the surface of mature monocytes andmacrophages in a soluble form. Native sCD14 lacks the GPI anchor and ispresent in serum. The biological origin and function of sCD14 have notyet been fully defined (Bazil, Europ. J. Immunol., 16:1583-1589, 1986).

As used herein, “soluble” is defined as not associated in the cellsurface. “Soluble CD14” is a non-cell-associated CD14 molecule furthercharacterized as specifically binding LPS:LBP complexes and/or LPSalone. “Recombinant human sCD14” includes both a full-length amino acidsoluble human CD14 protein encoded by the nucleic acid sequence in FIG.1 (SEQ ID NO:9) and its truncated version. For the purposes ofidentification only, the full-length protein is designated 523 and thetruncated version is designated 847. This human sCD14 is useful as animmunogen for the generation of polyclonal and monoclonal antibodies andto detect the presence of LPS in a patient sample. When used as animmunogen, 523 provided advantages over prior art CD14 immunogens, e.g.,523 provided a higher number of CD14specific positive clones, iteliminated the number of non-specific responses to other immunogenicproteins which would be present in whole cell extracts, and it decreasedthe number of screening attempts needed to obtain the antibodies ofinterest. This full length human sCD14 polypeptide has the nucleic acidsequence as shown in FIG. 1, SEQ ID NO:9. Using this sequence, one ofskill in the art can produce polypeptide of like sequence by chemicalsynthesis or recombinantly. The truncated version (“847”) has eight (8)amino acids from the carboxyl end of the sequence deleted.

Minor modifications of sCD14 primary amino acid sequence may result inproteins which have substantially equivalent function compared to thesCD14 protein described herein. Such modifications may be deliberate, asby site-directed mutagenesis, or may be spontaneous. All proteinsproduced by these modifications are included herein as long as sCD14function exists.

Modifications of sCD14 primary amino acid sequence also includeconservative variations. The term “conservative variation” as usedherein denotes the replacement of an amino acid residue by another,biologically similar residue. Examples of conservative variationsinclude the substitution of one hydrophobic residue such as isoleucine,valine, leucine or methionine for another, or the substitution of onepolar residue for another, such as the substitution of arginine forlysine, glutamic for aspartic acids, or glutamine for asparagine, andthe like. The term “conservative variation” also includes the use of asubstituted amino acid in place of an unsubstituted parent amino acidprovided that antibodies raised to the substituted polypeptide alsoimmunoreact with the unsubstituted polypeptide.

The invention provides a nucleic acid molecule encoding the humansoluble CD14 polypeptide as shown in FIG. 1 (SEQ ID NO:9). The inventionalso encompasses nucleic acids molecules which differ from that of thenucleic acid molecule shown in FIG. 1, but which produce the samephenotypic or immunogenic effect when the nucleic acid molecule isexpressed. This invention encompasses nucleic acid moleculescharacterized by changes in non-coding regions that do not alter thephenotype of the polypeptide produced therefrom when compared to thenucleic acid molecule described hereinabove. Therefore, it is understoodthat all polynucleotides encoding all or a portion of sCD14 are alsoincluded herein, so long as they exhibit a function of sCD14, such asthe ability to induce or bind antibody. Such polynucleotides includeboth naturally occurring and intentionally manipulated, for example,mutagenized polynucleotides. These polynucleotides include DNA and RNAsequences which encode the protein

This invention further encompasses nucleic acid molecules whichhybridize to the nucleic acid molecule of the subject invention. As usedherein, the term “nucleic acid” encompasses RNA as well as single anddouble stranded DNA and cDNA.

Using the sequence provided in FIG. 1 and methods well known to those ofskill in the art (as exemplified in Sambrook, et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989),incorporated throughout by reference), human sCD14 can be recombinantlyproduced and isolated. Expression vectors containing this sequence, aswell as host cells containing the vectors, are also provided by thisinvention. As used herein, the term “vector” or “expression vector”refers to sequences of heterologous nucleic acids which are capable ofbeing expressed in selected host cells through operational associationwith other sequences capable of effecting their expression, such aspromoter and enhancer sequences. For the purpose of illustration only,these expression vectors can be bacterial plasmids, bacterial phages,animal viruses, baculoviruses or cosmids. Procaryotic host cells such asE. coli can be used for recombinantly producing these polypeptides whenthe vector is a bacterial plasmid or a bacterial phage. Eucaryotic hostcells can be, but are not limited to mammalian host cells, e.g., ChineseHamster Ovary Cells (CHO) or insect cells for baculoviral expression.

A method of recombinantly producing the human sCD14 is provided by thisinvention. This method requires growing the host cells described aboveunder suitable conditions such that the sCD14 nucleic acid molecule istranscribed and translated. Upon expression, the recombinant sCD14 canbe isolated from the cell culture by use of an affinity column composedof commercially available CD14 monoclonal antibody.

This invention also provides polyclonal antibodies and monoclonalantibodies, specifically reactive with cell surface CD14 receptor andsoluble CD14. The antibodies of the invention inhibit CD14 mediated cellactivation by a ligand otherwise capable of binding to the CD14 receptorand activating the cell, for example, to induce NK-κB activation orproduce and release a cytokine. Monoclonal antibodies provided hereinare capable of inhibiting CD14 mediated cell activation by the ligandeven when the ligand has bound to CD14. The monoclonal antibodies mayallow at least about 50% ligand binding to occur between the ligand andCD14, although these antibodies can allow at least about 80% binding ofligand to CD14 to occur and still be capable of inhibiting CD14 mediatedcell activation.

As used herein, a “antibody or polyclonal antibody” means a protein thatis produced in response to immunization with an antigen or throughrecombinant cloning techniques. The term “monoclonal antibody” means animmunoglobulin derived from a single clone of cells. All monoclonalantibodies derived from the clone are chemically and structurallyidentical, and specific for a single antigenic determinant.

Laboratory methods for producing polygonal antibodies and monoclonalantibodies are known in the art (see, Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1988),incorporated herein by reference). The monoclonal antibodies of thisinvention can be biologically produced by introducing full length humanrecombinant polypeptide for soluble CD14 into an animal, e.g., a mouseor a rabbit. The antibody producing cells in the animal are isolated andfused with myeloma cells or heteromyeloma cells to produce hybrid cellsor hybridomas. Accordingly, the hybridoma cells producing the monoclonalantibodies of this invention also are provided. Monoclonal antibodiesproduced in this manner include, but are not limited to the monoclonalantibodies designated 18E12, 28C5, 23G4, 5G3, 4F2, 13A7, 10B7, and 26F3.The hybridoma cell lines 18E12, 28C5 and 23G4 have been deposited withthe American Type Culture Collection (ATCC) 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A., under the provisions of the BudapestTreaty on the International Deposit of Microorganisms for the Purposesof Patent Procedure 18E12 and 28C5 were deposited on May 27, 1993 andwere accorded ATCC accession numbers HB11363 and HB11364, respectively23G4 was deposited on May 25, 1994 and was accorded ATCC accessionnumber X. These deposits were made under the provisions of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and the Regulations thereunder(Budapest Treaty). This assures maintenance of viable cultures for 30years from the date of deposit. The organisms will be made available byATCC under the terms of the Budapest Treaty which assures permanent andunrestricted availability of the progeny of the culture to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 U.S.C. §122 and the Commissioner's rules pursuant thereto(including 37 C.F.R. §1.14 with particular reference to 886 OG 638)

The assignee of the present application has agreed that if the culturedeposit should die or be lost or destroyed when cultivated undersuitable conditions, it will be promptly replaced on notification with aviable specimen of the same culture. Availability of a deposited strainis not to be construed as a license to practice the invention incontravention of the rights granted under the authority of anygovernment in accordance with its patent laws.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the cell lines deposited,since the deposited embodiment is intended as a single illustration ofone aspect of the invention and any cell lines that are functionallyequivalent are within the scope of this invention. The deposit ofmaterial does not constitute an admission that the written descriptionherein contained is inadequate to enable the practice of any aspect ofthe invention, including the best mode thereof, nor is it to beconstrued as limiting the scope of the claims to the specificillustration that it represents.

Thus, using the unique full length recombinant protein for soluble CD14and the well known methods, one of skill in the art can produce andscreen the hybridoma cells and antibodies of this invention.

The monoclonal antibodies of this invention can be characterized asbeing able to specifically react with cell surface CD14 receptor andsoluble CD14 and inhibit CD14 mediated cell activation. Methods ofdetermining binding specificities are outlined below. In one embodiment,the monoclonal antibodies are further characterized as having a bindingaffinity for CD14 greater than the affinity of anti-CD14 antibody 3C10(available from the American Type Culture Collection). One suchmonoclonal antibody is the monoclonal antibody designated 28C5.Scatchard analysis of 28C5 binding to dihydroxyvitamin D3 induced THPIcells gave an affinity of 3×10⁻⁹M⁻¹. Monoclonal antibody 28C5 and 23G4,and antibodies of like specificity and affinity, are furthercharacterized as being able to inhibit activation and also inhibit CD14binding of the ligand which induces NF-κB activation. In addition, allof the monoclonal antibodies of the invention can be characterized bytheir ability to inhibit cytokine release from CD14+ cells when suchcells are contacted with the inducing ligand. As used herein, a cytokineshall include, but is not limited to TNF-α, IL-1, IL-6, and IL-8.

In an alternative embodiment, the monoclonal antibody 18E12 andmonoclonal antibodies of like specificity are further characterized ashaving the ability to inhibit CD14 mediated cell activation, but do notsignificantly inhibit CD14 binding (i.e., these antibodies allow CD14binding) with the ligand which is otherwise capable of inducing CD14mediated cell activation. Monoclonal antibodies with the specificity of18E12 will allow from at least about 50% to at least about 80% bindingto occur between the ligand and CD14.

The preferred monoclonal antibodies described herein, 18E12 and 23G4,bind to both human and baboon CD14, whereas, 28C5 does not bind baboonCD14.

Although LBP is the predominant serum protein involved in presentationof LPS to CD14, other serum proteins may also bind to LPS underappropriate conditions and facilitate LPS-CD14 interactions (Wright, S.D., et al., J. Expt. Med., 176:719-727, 1992). Regardless of whether LBPor other proteins predominate under physiologic conditions the effectsof the monoclonal antibodies 18E12, 23G4 or 28C5 are the same sincethese antibodies prevent the effects of LPS on NF-κB or cytokineproduction in the presence plasma (or serum).

This invention also provides biological active fragments of thepolyclonal and monoclonal antibodies described above. These “antibodyfragments” retain some ability to selectively bind with its antigen orreceptor. Such antibody fragments can include, but are not limited to:

-   (1) Fab, the fragment which contains a monovalent antigen-binding    fragment of an antibody molecule produced by digestion with the    enzyme papain to yield an intact light chain and a portion of one    heavy chain;-   (2) Fab′, the fragment of an antibody molecule obtained by treating    with pepsin, followed by reduction, to yield an intact light chain    and a portion of the heavy chain; two Fab′ fragments are obtained    per antibody molecule;-   (3) (Fab′)₂, the fragment of the antibody that is obtained by    treating with the enzyme pepsin without subsequent reduction;    F(ab′)₂ is a dimer of two Fab′ fragments held together by two    disulfide bonds;-   (4) Fv, defined as a genetically engineered fragment containing the    variable region of the light chain and the variable region of the    heavy chain expressed as two chains; and-   (5) Single chain antibody (“SCA”), defined as a genetically    engineered molecule containing the variable region of the light    chain, the variable region of the heavy chain, linked by a suitable    polypeptide linker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art, see for example,Harlow and Lane, supra.

Additional examples of “biologically active fragment” include antibodyfragments specifically including the CDRs of the antibodies as definedbelow. These CDR regions are identified in FIGS. 2 through 5 and 29-30,(SEQ ID NOS:1 through 8 and SEQ ID NOS:22-24). CDRs of these antibodiesare useful to generate CDR grafted antibodies as described below.Additional examples of “biologically active fragments” include fragmentsspecifically including the framework regions of the antibodies alsoidentified in FIGS. 2 through 5 and 29-30, (SEQ ID NOS:1 through 8 andSEQ ID NOS:22-24) The framework regions of the antibodies are useful asprimers for PCR amplification of the CDRs.

Also encompassed by this invention are proteins or polypeptides thathave been recombinantly produced, biochemically synthesized, chemicallysynthesized or chemically modified, that retain the ability to bind CD14cell surface receptor and soluble CD14 as well as inhibit CD14 mediatedcell activation by binding of activating ligand to CD14+ cells, of thecorresponding native polyclonal or monoclonal antibody. The ability tobind with an antigen or receptor is determined by antigen-binding assaysknown in the art such as antibody capture assays (see, for example,Harlow and Lane, supra).

Any of the above described antibodies or biologically active antibodyfragments can be used to generate CDR grafted and chimeric antibodies.

“CDR” or “complementarity determining region” or “hypervariable region”is defined as the amino acid sequences on the light and heavy chains ofan antibody which form the three-dimensional loop structure thatcontributes to the formation of the antigen binding site.

As used herein, the term “CDR grafted” antibody refers to an antibodyhaving an amino acid sequence in which at least parts of one or more CDRsequences in the light and/or variable domain have been replaced byanalogous parts of CDR sequences from an antibody having a differentbinding specificity for a given antigen or receptor.

As used herein, the terms “light chain variable region” and “heavy chainvariable region” refer to the regions or domains at the N-terminalportion of the light and heavy chains respectively which have a variedprimary amino acid sequence for each antibody. The variable region ofthe antibody consists of the amino terminal domain of the light andheavy chains as they fold together to form a three-dimensional bindingsite for an antibody.

The analogous CDR sequences are said to be “grafted” onto the substrateor recipient antibody. The “donor” antibody is the antibody providingthe CDR sequence, and the antibody receiving the substituted sequencesis the “substrate” antibody. One of skill in the art can readily producethese CDR grafted antibodies using the teachings provided herein incombination with methods well known in the art (see Borrebaeck, C. A.,Antibody Engineering: A Practical Guide, W.H. Freeman and Company, NewYork, 1992, incorporated throughout by reference).

This invention further provides chimeric antibodies of the abovedescribed antibodies or biologically active fragments. As used herein,the term “chimeric antibody” refers to an antibody in which the variableregions of antibodies derived from one species are combined with theconstant regions of antibodies derived from a different species.Chimeric antibodies are constructed by recombinant DNA technology, andare described in Shaw, et al., J. Immun., 138:4534 (1987), Sun, L. K, etal., Proc. Natl. Acad. Sci. USA, 84:214-218 (1987), for example.

Nucleic acid molecules encoding the antibodies, monoclonal antibodies,biologically active fragments, chimeric antibodies and CDR graftedantibodies described above also are provided by this invention. “Nucleicacid” is intended to include single and double stranded DNA, cDNA andRNA. These nucleic acid molecules can be operationally linked topromoter of RNA transcription. The invention also encompasses nucleicacids molecules which differ from that of the nucleic acid moleculesdescribed above, but which produce the same phenotypic effect. Theinvention encompasses nucleic acid molecules characterized by changes innon-coding regions that do not alter the phenotype of the polypeptideproduced therefrom when compared to the nucleic acid molecule describedhereinabove This invention further encompasses nucleic acid moleculeswhich hybridize to the nucleic acid molecule of the subject invention Asused herein, the term “nucleic acid” encompasses RNA as well as singleand double stranded DNA and cDNA.

In one embodiment, these nucleic acid molecules are inserted intoexpression vectors as noted above. The expression vectors can beinserted into suitable host cells. When the cells are induced to growunder conditions favoring transcription and translation of the insertednucleic acid sequence, a recombinant protein or polypeptide is producedwhich can then be isolated and used for diagnosis or therapy asdescribed below. Methods of recombinantly producing polypeptides andproteins are generally known (see Sambrook, et al., supra and Kreigler,M., Gene Transfer and Expression: A Laboratory Manual, W.H. Freeman andCompany, New York, 1990, each incorporated herein by reference).

Pharmaceutical compositions also are provided by this invention. Thesepharmaceutical compositions contain any of the above describedpolypeptides, fragments, antibodies, monoclonal antibodies, antibodyfragments, chimeric antibodies or CDR grafted antibodies, each alone orin combination with each other, and a pharmaceutically acceptablecarrier. As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.These pharmaceutical compositions are useful for diagnostic ortherapeutic purposes.

The monoclonal antibodies of the invention are suited for in vitro foruse, for example, in immunoassays in which they can be utilized inliquid phase or bound to a solid phase carrier. In addition, themonoclonal antibodies in these immunoassays can be detectably labeled invarious ways. Examples of types of immunoassays which can utilizemonoclonal antibodies of the invention are competitive andnon-competitive immunoassays in either a direct or indirect format.Examples of such immunoassays are the radioimmunoassay (RIA) and thesandwich (immunometric) assay. Detection of the antigens using themonoclonal antibodies of the invention can be done utilizingimmunoassays which are run in either the forward, reverse, orsimultaneous modes, including competition immunoassays andimmunohistochemical assays on physiological samples. Those of skill inthe art will know, or can readily discern, other immunoassay formatswithout undue experimentation.

The monoclonal antibodies of the invention can be bound to manydifferent carriers and used to detect CD14. Examples of well-knowncarriers include glass, polystyrene, polypropylene, polyethylene,dextran, nylon, amyloses, natural and modified celluloses,polyacrylamides, agaroses, and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingmonoclonal antibodies, or will be able to ascertain such, using routineexperimentation.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds, andbio-luminescent compounds. Those of ordinary skill in the art will knowof other suitable labels for binding to the monoclonal antibodies of theinvention, or will be able to ascertain such, using routineexperimentation. Furthermore, the binding of these labels to themonoclonal antibodies of the invention can be done using standardtechniques common to those of ordinary skill in the art.

Further provided herein is a method of blocking the binding of LPS/LBPcomplex to a CD14 receptor on the surface of a cell by contacting thecell with a monoclonal antibody capable of binding to the complex, e.g.,a monoclonal antibody with the specificity and affinity of 28C5, or 23G4or a biologically active fragment of the monoclonal antibody. Alsodisclosed is a method of inhibiting NF-κB activation of a cellexpressing CD14 receptor in the presence of a ligand (such as LPS orLPS/LBP) which is capable of inducing NF-κB activation. This methodprovides contacting the cell with an antibody having the ability tospecifically bind cell surface receptor CD14 and full length humansoluble CD14 or a biologically active fragment of the antibody. Specificexamples of such antibodies are the antibodies designated 28C5, 23G4 and18E12.

Several therapeutic methods are provided herein which can be utilized inanimals and humans. One therapeutic method is a means to treat orprevent disease associated with NF-κB activation, such as sepsis, byadministering to a subject with the disease an effective amount of anantibody having the ability to specifically bind cell surface receptorCD14 and soluble CD14 or a biologically active fragment of the antibody.The above method is especially advantageous when a monoclonal antibodyhaving the binding specificity of 18E12 is used. Because 18E12 and thelike antibodies can inhibit NF-κB activation even after LPS has boundthe CD14 receptor, such antibody can be used for the treatment of laterstage sepsis. As used herein, later stage sepsis means the diseasecourse after LPS has bound CD14 cell-associated receptor. Significantly,18E12 and like antibodies are capable of allowing the cell to which theantibody has bound to continue to transport LPS or LPS/LBP complex intothe cell. This property provides the added benefit of allowing theremoval of LPS or LPS/LBP complex from the in vivo system therebyinhibiting the possible pathological interaction of LPS or LPS/LBPcomplex at some other in vivo site.

Alternatively, monoclonal antibodies 28C5 and 23G4 are preferred in themethod of the invention where treatment is prophylactic or it isdesirable to block LPS/LBP from binding to CD14, thereby inhibitingcytokine release and cell activation.

The invention provides a therapeutic method of ameliorating sepsis orone or more of the symptoms of sepsis comprising administering to asubject displaying symptoms of sepsis or at risk for developing sepsis,a therapeutically effective amount of a monoclonal antibody of theinvention that binds to CD14 and inhibits cell activation. Such symptomswhich may be ameliorated include those associated with a transientincrease in the blood level of TNF, such as fever, hypotension,neutropenia, leukopenia, thrombocytopenia, disseminated intravascularcoagulation, adult respiratory distress syndrome, shock and multipleorgan failure. Patients who require such treatment include those at riskfor or those suffering from toxemia, such as endotoxemia resulting froma gram-negative bacterial infection, venom poisoning, or hepaticfailure, for example. In addition, patients having a gram-positivebacterial, viral or fungal infection may display symptoms of sepsis andmay benefit from such a therapeutic method as described herein. Thosepatients who are more particularly able to benefit from the method ofthe invention are those suffering from infection by E. coli, Haemophilusinfluenza B, Neisseria meningitides, staphylococci, or pneumococci.Patients at risk for sepsis include those suffering from burns, gunshotwounds, renal or hepatic failure.

The term “therapeutically effective amount” as used herein refers to theamount of monoclonal antibody which binds to CD14 and blocks signallingevents such as cytokine release, used is of sufficient quantity todecrease the subject's response to LPS and decrease the symptoms ofsepsis. The term “therapeutically effective” therefore includes thatamount of antibody sufficient to prevent, and preferably reduce by atleast 50%, and more prererably sufficient to reduce by 90%, a clinicallysionificant increase in the plasma level of TNF, for example. The dosageranges for the administration of the monoclonal antibody of theinvention, for example 18E12, 28C5 and 23G4, are those large enough toproduce the desired effect. Generally, the dosage will vary with theage, condition, sex, and extent of the infection with bacteria or otheragent as described above, in the patient and can be determined by oneskilled in the art. The dosage can be adjusted by the individualphysician in the event of any contraindications. In any event, theeffectiveness of treatment can be determined by monitoring the level ofLPS and TNF in a patient. A decrease in serum LPS and TNF levels shouldcorrelate with recovery of the patient.

In addition, patients at risk for or exhibiting the symptoms of sepsiscan be treated by the method as described above, further comprisingadministering, substantially simultaneously with the therapeuticadministration of a monoclonal antibody of the invention, an inhibitorof TNF, an antibiotic, or both. For example, intervention in the role ofTNF in sepsis, either directly or indirectly, such as by use of ananti-TNF antibody and/or a TNF antagonist, can prevent or ameliorate thesymptoms of sepsis. Particularly preferred is the use of an anti-TNFantibody as an active ingredient, such as a monoclonal antibody with TNFspecificity as described by Tracey, et al. (Nature, 330:662, 1987).

A patient who exhibits the symptoms of sepsis may be treated with anantibiotic in addition to the treatment with a truncated LBP or antibodyof the invention. Typical antibiotics include an aminoglycoside, such asgentamycin or a beta-lactam such as penicillin, or cephalosporin.Therefore, a preferred therapeutic method of the invention includesadministering a therapeutically effective amount of an antibody of theinvention, substantially simultaneously with administration of abactericidal amount of an antibiotic.

The term “bactericidal amount” as used herein refers to an amountsufficient to achieve a bacteria-killing blood concentration in thepatient receiving the treatment. The bactericidal amount of antibioticgenerally recognized as safe for administration to a human is well knownin the art, and as is known in the art, varies with the specificantibiotic and the type of bacterial infection being treated.

Preferably, administration of a monoclonal antibody of the inventionoccurs within about 48 hours and preferably within about 2-8 hours, andmost preferably, substantially concurrently with administration of theantibiotic.

For the purposes of this invention, a subject is an animal or a humanpatient and an effective amount is from about 0.25 mg/kg/body weight toabout 50 mg/kg/body weight. In one embodiment, the effective amount isfrom about 0.5 mg/kg/body weight to about 10 mg/kg/body weight. When thesubject is a human patient, the preferred amount is from about 0.5mg/kg/body weight to about 8 mg/kg/body weight.

As is known to those of skill in the art, the above methods may becombined to enhance the therapeutic and prophylactic effects. Means ofadministering pharmaceutical compositions are well known to those ofskill in the art and include, but are not limited to administrationintravenously, orally, intraperitoneally, subcutaneously or byinhalation therapy.

The following examples are intended to illustrate but not limit theinvention.

EXAMPLE 1 Generation of Soluble CD14 (sCD14) and Production ofMonoclonal Antibodies

A. Cloning of sCD14

A copy of the human CD14 gene was obtained. The description of thecloning of this gene, from a human monocytic cell line (HL-60) (AmericanType Culture Collection, ATCC No. 240), is described in Blood, 73:284(1989), incorporated herein by reference. The CD14 gene was excised fromthis expression vector and cloned into the mammalian expression vectorpEE14 (Celltech). This vector has an inducible glutamine synthetase gene(GS) which was used to amplify the inserted DNA fragments containing theCD14 gene. A full-length DNA sequence of the gene was cloned into pEE14.Cells expressing soluble CD14 were identified as an ELISA assay byreactivity with commercially available anti-CD14 mAbs. One clone,identified as 523, was demonstrated to express both soluble CD14 and amembrane associated form which could be detected by FACS analysis. Thesoluble form of clone 523 was determined to be N-terminally processed atamino acid residue 20 of the predicted translated protein sequence. Thesequence for this protein is set forth in FIG. 1. Amino acid residues1-19 of the translated CD14 sequence was predicted to be a signalsequence (Gene Works, Intelligenetics). It was determined by C-Terminalsequence analysis that the C-terminus was intact; no processing hadoccurred which was similar to that noted in the soluble CD14 isolatedfrom human serum (Bazil, et al., Eur. J. Immunol., 16:1583, 1986,incorporated herein by reference). The soluble CD14 isolated from urineof nephritic patients is lacking the eight most C-terminal amino acids(Bazil, Mol. Immunol., 26:657, 1989). The clone 523 may have avoided theprocessing steps at the C-terminus as a consequence of its expression inCHO cells.

Purification of the soluble CD14 was accomplished by purifying theantigen on an affinity column composed of commercially available mAb63D3 (obtainable from the American Type Culture Collection (ATCC No. HB44)).

B. Generation of sCD14 Monoclonal Antibodies

Monoclonal antibodies to human soluble CD14 were generated by somaticcell fusion between spleen cells from BALB/c mice immunized withpurified human recombinants CD14 described above, and the mouse myelomacell line X63.Ag8.653. Monoclonal antibodies 28C5, 18E12, 26F3 and 23G4are IgG1 mAb which were identified by screening against CD14 in an ELISAassay. Binding to native CD14 was confirmed by flow microfluoremetry onCD14⁺ cells and immunoprecipitation of biosynthetically labeled CD14.Monoclonal antibodies 28C5, 23G4 and 18E12 recognize cell-associated andsoluble CD14. Competition studies indicated that these mAb bound tothree distinct CD14 epitopes (overlap between 28C5 and 23G4).

EXAMPLE 2 Characterization of Monoclonal Antibodies

A. FACS Analysis of CD14⁺ Positive Cells with Anti-CD14 MonoclonalAntibodies

THP-1 cells (American Type Culture Collection, ATCC No. TIB 202) wereinduced with dihydroxyvitamin D3 for 48 hours and cells were then washedwith DMEM (Dulbecco's modified Eagles medium with 4.5 g/L glucose, 90%fetal bovine serum, 10% followed by PBS with 1% BSA and 0.02% azide).One million cells/tube were reacted with first antibody (anti-CD14ssupernatants) at 1:2 dilution for 45 minutes at 4° C. Cells are washedwith PBS/BSA/azide. The second antibody was added (goat anti-mouse IgG,FITC-labeled (Cappel)) at a 1:250 dilution for 30 minutes at 4° C. Cellsare washed 2× with same buffer. Pellets resuspended in 1 ml of buffer.Fluorescence intensity was measured by a Cytofluorograph (OrthoInstruments). The results are shown in FIGS. 6 through 10.

B. Determination of Binding Affinity of Anti-sCD14 Monoclonal Antibodiesfor Soluble CD14

All anti-CD14 mAbs, and some commercially available anti-CD14s, wereevaluated for antibody affinity to soluble CD14 antigen (FIG. 11).Antibody 3C10 had a titer of 1:2⁸ and 28CS a titer of 1:2¹¹. This showsa eight-fold difference in titer of 28C5 as compared to 3C10. Relativeaffinities of the antibodies were determined at equivalentconcentrations of purified antibody protein which were probed with alabeled goat anti-mouse conjugated antibody. Those antibodies with thehighest affinity for sCD14, presented in this manner, are 4F2, 5G3,26F3, 28C5, 23G4 and 63D3. Anti-CD14 mAbs 3C10 and 18E12 exhibited thelowest affinity for sCD14. Anti-CD14 28C5 had a much higher affinity ascompared to 3C10. Soluble CD14 was coated onto microtiter plates and theanti-CD14 mAbs added in two-fold serial dilutions starting with 2 μg/ml.A goat anti-mouse HRP conjugated antibody is added and the platesincubated and washed. Substrate is then added and the 1:2^(n) titer isrecorded. This is represented by the highest dilution of antibody givingat least 3× the OD_(490nm) value of a negative control.

Competition studies between labeled 3C10 and a panel of anti-CD14 mAbsrevealed that only antibodies 28C5 and 10A1 (an I_(g)A mMab) werecapable of competing with 3C10 for sCD14 on the coated microtiter plate(FIG. 12). Further competition assays were performed and confirmed that18E12 did not compete with 28C5, 3C10, or 26F3.

Table 1 shows the results of a similar competition assay between 28C5,23G4 and 18E12. The antibodies were coated on the solid phase in anELISA assay with biotin labeled anti-CD14 mAbs (A,C). TABLE 1COMPETITION STUDIES BETWEEN DIFFERENT ANTI-CD14 MABS FOR CD14RECOGNITION AND THE ABILITY OF THESE MABS TO BLOCK LPS/LBP BINDING TOCD14 A +18E12 B +28C5 C +23G4 D LPS Binding Antibody % Inhibition %inhibition % inhibition % inhibition 18E12 95.5 59.8 30.4 3.6 28C5 46.190.6 69.7 83.3 23G4 77.3 95.5 95.5 85.4

The ability of these anti-CD14 mAbs to block LPS/LBP binding to sCD14was assessed using as similar ELISA format.

When the mAbs were evaluated for ability to inhibit LPS/LBP binding tosCD14, 28C5 was the most effective (FIG. 13). FIG. 13 represents theintensity of binding of LPB/biotinylated LPS complex to soluble CD14immobilized on a solid phase, in the presence of 4 μg/ml of differentanti-CD14 monoclonal antibodies. Anti-CD14 mAb 28C5 and 3C10 block thisbinding event. Anti-CD14 mAb is more efficient in its blocking as notedby the decreased OD value. Anti-CD14 mAb 18E12 does not exhibit anyblocking effect.

Evaluation of the anti-CD14 mAbs for the ability to block cytokinerelease in HL-60 cells in response to LPS stimulation, showed that 28C5blocked TNF-α expression (FIG. 14). Inhibition of cytokine release wasalso observed when 28C5, 23G4, and 18E12 were added to whole blood exvivo prior to addition of LPS. Surprisingly, 18E12 inhibited cytokinerelease even though it was previously demonstrated not to block LPS/LBPbinding to CD14 (FIG. 13 and 27). The effect of 23G4, 28C5 and 18E12 onLPS-induced TNF in baboon whole blood was also examined, ex vivo. Theresults in FIG. 28 show that 23G4 was most effective at inhibiting TNFsecretion in LPS-induced baboon blood. These results show that 18E12 wasspecific for a domain on CD14 which did not prevent LPS/LBP binding, butis important to the signaling events involved in LPS stimulation ofcells.

Although 28C5 and 23G4 share specificity in blocking binding of LPS:LBPto CD14, they do not share recognition of baboon CD14 (only 23G4recognizes baboon), nor can 28C5 block TNFα release from baboon wholeblood in response to LPS (FIG. 27).

The ability of the anti-CD14 mAbs, 28C5, 18E12 and 23G4 to block LPS/LBPbinding to sCD14 was also assessed using a similar ELISA format asdescribed for Table 1. The results also show that 23G4 and 28C5 competefor sCD14 binding (see Table 1).

C. Activation of Cytokine Release

HL-60 cells (obtained from the American Type Culture Collection, ATCCNo. CCL-240 were plated at a concentration of 1.5×10⁵ cells per ml. Thecells were induced toward the monocyte lineage for 3 to 4 days in RPMI1640 containing 10% bovine serum, 10⁻⁷ M DHvD3 (Biomol ResearchLaboratories) and 50 μM indomethacin (Calbiochem). These differentiatedcells were resuspended at 1×10⁶ cells/ml growth medium containing 50 μMindomethacin with or without 10% human type AB serum (Irvine Scientific)and then were added to flat bottom cluster dishes. Cells were activatedby addition of different concentrations of LPS (E. coli serotype01217:B8; Sigma) followed by a 4 to 5 hour incubation at 37° C. Cells inthe culture plates were pelleted by low speed centrifugation (170× g for10 minutes at room temperature) and the growth medium was removed forELISA (ELISA kit for human TNF-α detection; Genzyme) of soluble cytokinelevels.

D. Inhibition of LPS Binding to Cellular CD14 by Anti-CD14 MonoclonalAntibodies

To characterize the mechanism of interaction between cellular CD14 andLPS, stably transfected 70Z/3 cells containing the human CD14 expressionvector described in Lee, et al., J. Exp. Med., 175.1697-1705, 1992, wereprepared to form 70Z/3-hCD14 cells. Stably transfected cells expressingcell surface CD14 were confirmed using FACS analysis on cells stainedwith FITC conjugated anti-human CD14 Mab MY4 described earlier. Othertransfected cell lines were also prepared expressing CD14 fusionproteins in which the membrane anchor from CD14 was removed and replacedwith the membrane anchor from decay accelerating factor (DAF),designated 70Z/3-hCD14DAF, with the membrane anchor from human tissuefactor, designated 70Z/3-hCD14TF, and with the membrane anchor from themurine class molecule, H2K², designated 70Z/3-hCD14Cl.

Direct binding of LPS to cellular CD14 was characterized usingFITC-labelled LPS (FITC-LPS). 70Z/3-CD14 cells were suspended in culturemedium containing 10% FCS with or without 10 ug/ml Mab, andpre-incubated for 30 minutes at 37° C. Thereafter, FITC-Re595-LPS wasadded at 1 ng/ml and maintained for 15 minutes at 37° C. Immediatelythereafter, an equal volume of ice-cold RPMI 1640 medium was added, andthe admixture was maintained at 4° C. until FACS analysis.Cell-associated fluorescence was measured as described by Lee et al.,supra, and measured fluorescence adjusted by subtracting fluorescencemeasured using non-transfected 70Z/3 cells.

The bar-graph results are shown in FIG. 15, and indicate that for alltransfected cell types that contain a cell-surface CD14 protein (wt orfusion protein), the anti-CD14 monoclonal antibody Mab MY4 (shaded bar)blocked LPS binding to cells, whereas Mab 18E12 (open bar) did not blockLPS binding to cells. Binding of FITC-LPS in the presence of Mab 18E12was similar to results obtained using no antibody (black bar). Mab MY4is an antibody known to immunoreact with CD14, and by the data presentedherein is shown to inhibit LPS binding to CD14 and to inhibitLPS-dependent, CD14-mediated cell activation. The differences in levelsof FITC-LPS binding reflect the differences in levels of CD14 expressionin the different transfected cell lines. Whereas the hCD14 transfectedcells contain approximately 10,000 receptors per cell, the hCD14DAFtransfected cells contain approximately 50,000 receptors per cell, andthe hCD14Tf and hCD14Cl transfected cells are estimated to each containabout 15,000-20,000 receptors per cell. The results are expressed as amean±standard deviation of three independent determinations.

E. Inhibition of LPS-Dependent, CD14-Mediated Activation of Cells UsingAnti-CD14 Monoclonal Antibodies

Anti-CD14 monoclonal antibodies were characterized for their ability toinhibit CD14-mediated activation of cells by LPS. To that end, a CD14transfected cell system was developed and demonstrated to be responsiveto LPS-induced activation. Several transfected 70Z/3 cell lines wereprepared as described in Example 3, and contain several membraneassociated forms of CD14 as described earlier.

The transfected cells were cultured as described by Lee et al., supra,suspended in RPMI 1640 media containing 10% fetal calf serum (FCS; heatinactivated, 56° C. for 30 min.) and 10 ug/ml antibody (MY4 or 18E12) asindicated by a “+” in FIG. 16, and maintained for 30 min at 37° C.Thereafter, 100 uM taxol or LPS (1 ng/ml Re595 LPS) was added asindicated by a “+” in FIG. 16 and the cells were maintained for 15 minat 37° C. Thereafter, the cells were harvested and nuclear extracts wasprepared to determine activation of NF-κB as described by Molitor etal., Proc. Natl. Acad. Sci. USA, 87:10028-10032 (1990). P³²-labelledNF-κB-specific oligonucleotides (5′-CAGAGGGGACTTTCCGAGA-3′) indouble-stranded form were used in a gel retardation assay to detect thepresence of NF-κB on 4% non-denaturing polyacrylamide gels.

The results of the study are shown in FIG. 16, and indicate that LPS andtaxol both induce NF-κB activation. As expected, LPS induced minimalNF-κB activation in control transfected cells lacking CD14 (70Z/3-RSV),and induced marked activation in cells expressing CD14. This shows thatLPS-induced NF-κB activation is mediated by and requires CD14 on thecell surface. Furthermore, the results show that both MY4 and 18E12inhibit LPS-induced NF-κB expression, but not taxol-induced NF-κBexpression, indicating that the inhibitory effect of the antibodies isspecific and dependent upon CD14.

These results with Mabs MY4 and 18E12 indicate that LPS binding to CD14is not sufficient to induce cell activation and that additionalinteractions following LPS-CD14 binding are critical for cellactivation. The results also indicate that inhibition of CD14-mediatedactivation of cells may occur at different levels, first by blocking theinducer (LPS) from binding to CD14, and second by blocking a subsequentstep after inducer binds to CD14. The data also establish that the useof inhibitors of the second step will block CD14-mediated cellactivation where the inducer is a molecule other than LPS.

F. Inhibition of LPS Uptake by CD14⁺ Cells

In the progression of sepsis, LPS binds cell surface CD14, and is knownto be taken up by those CD14⁻ cells (Kitchens, et al., J. Exp. Med.,176:485-494, 1992; Pugin, et al., PNAS, 90:2744-2748, 1993). Thedifference between antibodies that block LPS binding to CD14, such asMab 28C5, and antibodies that do not block binding to CD14, such as Mab18E12, is significant in view of LPS uptake by CD14⁻ cells. Therefore,the ability of anti-CD14 antibodies to inhibit LPS uptake wascharacterized.

To that end, FITC-LPS uptake on transfected 70Z/3-hCD14 cells wasmeasured in the presence of various anti-CD14 antibodies. Fluorescencearising from the FITC-LPS was detected inside CD14⁺ transfected cellswhen no antibody was utilized after the cells were maintained at 37° C.following exposure to FITC-LPS, confirming that LPS is taken up by cellsunder normal conditions. In the presence of Mab 28C5, uptake of LPScould be inhibited completely, whereas in the presence of Mab 18E12,uptake could only be reduced to about 65% of the amount of uptakeobserved under normal conditions. These results indicate that Mab 18E12is particularly useful for inhibiting CD14⁺ cell activation where it isdesirable to allow LPS to enter the cells, because Mab 18E12 does notsubstantially prevent LPS uptake. Subsequent studies show that in thepresence of Mab 23G4, uptake of LPS could be inhibited similar to thatseen with Mab 28C5.

CD14 Antigen Assay (ELISA)

-   Coating: 150 μl/well of anti-CD14 mAb 28C5 diluted at 1 μg/ml in    bicarbonate buffer. Incubate overnight at 4° C.-   Blocking: Wash the plate 4× then add 150 μl/well of blocking buffer.    Incubate 1 hr. at 37° C.-   Samples: Wash the plate 1× then add 125 μl/well of samples diluted    in dilution buffer. Incubate 1 hr. at 37° C.-   Conjugate: Wash the plate 5× then add 0.100 ml/well of biotinylated    anti-CD14 mAb 18E12 diluted at 1 μg/ml in dilution buffer. Incubate    1 hr. at 37° C.-   Av-HRPO: Wash the plate 5× then add 0.100 ml/well of preformed    streptavidin/biotin/peroxidase complex.    (Streptavidin/bio-tinylated/HRPO preparation (Zymed SABC kit): Mix 2    μl/ml of Streptavidin with 2 μl/ml of biotinylated-HRPO in washing    buffer and incubate 30 minutes at 37° C. Before adding to the wells,    dilute at 1:2 with dilution buffer.) Incubate 30 minutes at 37° C.-   Substrate: Wash the plate 5× then add 0.100 ml/well of Sigma OPD,    leave the plate 30 minutes in the dark and stop the color    development with 0.050 ml of 4N H2SO4. Read plate at 490 nm.-   CD14 Standard: 2-fold serial dilutions of clone 523 at 100 ng/ml.-   Serum Dilutions: Starting dilution 1:25-1:50.

Miscellaneous ELISA Reagents

-   Blocking Buffer: PBS+10% w/v of nonfat dry milk (Carnation).-   Washing Buffer: PBS+0.05% v/v of Tween 20.-   Dilution Buffer: Mix vol/vol blocking buffer and wash buffer, use to    dilute samples, labelled antibody and the preformed complex.

EXAMPLE 3 Monoclonal Antibody Cloning

Messenger RNA was extracted from monoclonal antibody producing celllines using the method of Chomczynkski and Sacchi, Anal. Bio.,162:156-159 (1987), incorporated herein by reference. Reversetranscription was performed using murine specific 3″ antibody primers(IgG1 or k) and the resulting cDNAs subjected to PCR (Supplier)according to the manufacturer's instructions, using a panel of murinespecific 5′ antibody primers described in Huse, et al., Science,246:1275-1281 (1989), incorporated herein by reference. Heavy and lightchain DNA fragments were gel purified and digested with appropriateenzymes. The 672 base pair heavy chain fragment was cloned into theSpe1/Xho1 site of pBluescript II KS⁺ and sequenced using the automatedABI Model 373A DNA sequencer, according to the manufacturer'sinstructions. The 642 base pair light chain fragment was cloned into theSst1/Xba of pBluescript II KS⁺ and sequenced in a similar manner.

SEQ ID NO:1 and 2 are the nucleotide and deduced amino acid sequence ofthe 28C5 heavy chain and SEQ ID NO:3 and 4 are the nucleotide anddeduced amino acid sequence of the 28C5 light chain. SEQ ID NO:5 and 6are the the nucleotide and deduced amino acid sequence of the 18E12heavy chain and SEQ ID NO:7 and 8 are the nucleotide and deduced aminoacid sequence of the 18E12 light chain (FIGS. 2-5). FIG. 30 shows acomparison of the heavy chains of 3C10, 28C5 and 18E12. SEQ ID NO:25 and26 are the nucleotide and deduced amino acid sequence of the 23G4 lightchain (See FIG. 29). FIG. 29 shows the amino acid sequence of the lightchains of monoclonal antibodies 3C10, 28C5, 23G4 and 18E12. FIG. 30shows the amino acid sequence of the heavy chains of monoclonalantibodies 3C10, 28C5, and 18E12.

Note that although both 28C5 and 23G4 share the same specificity in thatthey block LPS:LBP binding to CD14, compete with each other for sCD14binding and prevent TNFα release in human whole blood at similarconcentrations (see FIG. 27), their light chains do not share the samenucleotide and amino acid sequence. (See Table 2)

Recombinant Expression of Nucleic Acids

The recombinant expression of nucleic acids of this invention areperformed according to the following general strategy. PolyA⁻ mRNA isisolated from the antibody-expressing hybridoma cells. cDNA synthesisand PCR amplification of the mRNA are performed by methods describedabove. From the cDNA sequence data obtained, the amino acid sequences ofthe polypeptides encoded by the DNA sequences are deduced by a computersoftware program, for example, MAPSEQ, commercially available fromDNAStar (Madison, Wis.).

The expression products, assembled as an antibody fragment, are screenedfor binding affinity by methods known in the art such as ELISAs(Enzyme-Linked Immuno-Sorbent Assay) utilizing the hapten or antigen, oraffinity columns (as described, for example, in Skerra and Pluckthun,Science, 240:1038-1041, 1988, incorporated herein by reference).

Several types of vectors are available and can be used to practice thisinvention, e.g., plasmid, DNA and RNA viral vectors, baculoviralvectors, and vectors for use in yeast. When the vector is a plasmid, itgenerally contains a variety of components including promoters, signalsequences, phenotypic selection genes, origin of replication sites, andother necessary components as are known to those of skill in the art.

Promoters most commonly used in prokaryotic vectors include the lac Zpromoter system, the alkaline phosphatase pho A promoter, thebacteriophage λPL promoter (a temperature sensitive promoter), the tacpromoter (a hybrid trp-lac promoter that is regulated by the lacrepressor), the tryptophan promoter, and the bacteriophage T7 promoter.

Promoters used to practice this invention are the lac Z promoter and thepho A promoter. The lac Z promoter is regulated by the lac repressorprotein lac i, and thus transcription of the polypeptide can becontrolled by manipulation of the level of the lac repressor protein. Byway of illustration, a phagemid containing the lac Z promoter is grownin a cell strain that contains a copy of the lac i repressor gene, arepressor for the lac Z promoter. Exemplary cell strains containing thelac i gene include JM 101 and XL1-blue. In the alternative, the hostcell can be cotransfected with a plasmid containing both the repressorlac i and the lac Z promoter. Occasionally both of the above techniquesare used simultaneously, that is, phagmid particles containing the lac Zpromoter are grown in cell strains containing the lac i gene and thecell strains are cotransfected with a plasmid containing both the lac Zand lac i genes. Normally when one wishes to express a gene, to thetransfected host above, one would add an inducer such asisopropylthiogalactoside (IPTG), but this step can be omitted.

Another useful component of vectors used to practice this invention is asignal sequence. This sequence is typically located immediately 5′ tothe gene encoding the polypeptide, and thus will be transcribed at theamino terminus of the fusion protein. However, in certain cases, thesignal sequence has been demonstrated to be located at positions otherthan 5′ to the gene encoding the protein to be secreted. This sequencetargets the protein to which it is attached across the inner membrane ofthe bacterial cell. The DNA encoding the signal sequence can be obtainedas a restriction endonuclease fragment from any gene encoding a proteinthat has a signal sequence. Suitable prokaryotic signal sequences can beobtained from genes encoding, for example, LamB or OmpF (Wong, et al.,Gene, 68:193, 1983, incorporated herein by reference), MalE, PhoA, OmpAand other genes. A preferred prokaryotic signal sequence for practicingthis invention is the E. coli heat-stable enterotoxin II (STII) signalsequence as described by Chang, et al., Gene, 55:189, 1987, incorporatedherein by reference.

Another useful component of the vectors used to practice this inventionis a phenotypic selection gene. Typical phenotypic selection genes arethose encoding proteins that confer antibiotic resistance upon the hostcell. By way of illustration, the ampicillin resistance gene (amp), andthe tetracycline resistance gene (tet) are readily employed for thispurpose.

Construction of suitable vectors comprising the aforementionedcomponents as well as the gene encoding the desired polypeptide areprepared using standard recombinant DNA procedures. References forrecombinant methodology have been provided infra. Isolated DNA fragmentsto be combined to form the vector are cleaved, tailored, and ligatedtogether in a specific order and orientation to generate the desiredvector.

The DNA is cleaved using the appropriate restriction enzyme or enzymesin a suitable buffer. In general, about 0.2-1 μg of plasmid or DNAfragments is used with about 1-2 units of the appropriate restrictionenzyme in about 20 μl of buffer solution. Appropriate buffers, DNAconcentrations, and incubation times and temperatures are specified bythe manufacturers of the restriction enzymes. Generally, incubationtimes of about one or two hours at 37° C. are adequate, although severalenzymes require higher temperatures. After incubation, the enzymes andother contaminants are removed by extraction of the digestion solutionwith a mixture of phenol and chloroform and the DNA is recovered fromthe aqueous fraction by precipitation with ethanol.

To ligate the DNA fragments together to form a functional vector, theends of the DNA fragments must be compatible with each other. In somecases, the ends will be directly compatible after endonucleasedigestion. However, it may be necessary to first convert the sticky endscommonly produced by endonuclease digestion to blunt ends to make themcompatible for ligation. To blunt the ends, the DNA is treated in asuitable buffer for at least 15 minutes at 15° C. with 10 units of theKlenow fragment of DNA poiymerase I (Klenow) in the presence of the fourdeoxynucleotide triphosphates. The DNA is then purified byphenol-chloroform extraction and ethanol precipitation.

The cleaved DNA fragments are size-separated and selected using DNA gelelectrophoresis. The DNA is electrophoresed through either an agarose ora polyacrylamide matrix. The selection of the matrix will depend on thesize of the DNA fragments to be separated. After electrophoresis, theDNA is extracted from the matrix by electroelution, or, if low-meltingagarose is used as the matrix, by melting the agarose and extracting theDNA from it.

The DNA fragments that are to be ligated together (previously digestedwith the appropriate restriction enzymes such that the ends of eachfragment to be ligated are compatible) are put in solution in aboutequimolar amounts. The solution will also contain ATP, ligase buffer anda ligase such as T4 DNA ligase at about 10 units per 0.5 μg of DNA. Ifthe DNA fragment is to be ligated into a vector, the vector is at firstlinearized by cutting with the appropriate restriction endonuclease(s).The linearized vector can then be treated with alkaline phosphatase orcalf intestinal phosphatase. The phosphatasing prevents self-ligation ofthe vector during the ligation step.

After ligation, the vector with the foreign gene now inserted istransformed into a suitable host cell. Suitable prokaryotic host cellsinclude E. coli strain JM101, E. coli K12 strain 294 (ATCC number31,446), E. coli strain W3110 (ATCC number 27,325), E. coli X1776 (ATCCnumber 31,537), E. coli XL-1Blue (Stratagene), and E. coli B; however,many other strains of E. coli, such as HB101, NM522, NM538, NM539 andmany other species and genera of prokaryotes can be used as well. Inaddition to the E. coli strains listed above, bacilli such as Bacillussubtillis, other enterobacteriaceae such as Salmonella typhimunium orSerratia marcesans and various Pseudomonas species can all be used ashosts.

Transformation of prokaryotic cells is readily accomplished usingcalcium chloride or other methods well known to those skilled in theart. Electroporation (Neumann, et al., EMBO J., 1:841 1982, incorporatedherein by reference) also can be used to transform these cells. Thetransformed cells are selected by growth on an antibiotic, commonlytetracycline (tet) or ampicillin (amp), to which they are renderedresistant due to the presence of tet and/or amp resistance genes on thevector.

After selection of the transformed cells, these cells are grown inculture and the plasmid DNA (or other vector with the foreign geneinserted) is then isolated. Plasmid DNA can be isolated using methodsknown in the art. This purified plasmid DNA is then analyzed byrestriction mapping and/or DNA sequencing.

Following procedures outlined above, mammalian cell lines such asmyeloma (P3-653), hybridoma (SP2/0), Chinese Hamster Ovary (CHO), Greenmonkey kidney (COS1) and murine fibroblasts (L492) are suitable hostcells for polypeptide expression. These “mammalian” vectors can includea promoter, an enhancer, a polyadenylation signal, signal sequences andgenes encoding selectable markers such as geneticin (neomycinresistance), mycophenolic acid (xanthine guanine phosphoribosyltransferase) or histidinol (histidinol dehydrogenase).

Suitable promoters for use in mammalian host cells include, but are notlimited to, Ig Kappa, Ig Gamma, cytomegalovirus (CMV) immediate early,Rous Sarcoma Virus (RSV), simian virus 40 (SV40) early, mouse mammarytumor (MMTV) virus and metallothionein. Suitable enhancers include, butare not limited to Ig Kappa, Ig Heavy, CMV early and SV40. Suitablepolyadenylation sequences include Ig Kappa, Ig Gamma or SV40 large Tantigen. Suitable signal sequences include Ig Kappa, Ig Heavy and humangrowth hormone (HGH).

When the vector is baculovirus, suitable promoters and enhancersequences include, but are not limited to AcMNPV polyhedrin, AcMNPV ETLand AcMNPV p10 sequences. One particularly suitable polyadenylationsignal is the polyhedrin AcMNPV. Ig Kappa, Ig Heavy and AcMNPV areexamples of suitable signal sequences. These vectors are useful in thefollowing insect cell lines, among others: SF9, SF21 and High 5.

Alternatively, the polypeptides can be expressed in yeast strains suchas PS23-6A, W301-18A, LL20, D234-3, INVSC1, INVSC2, YJJ337. Promoter andenhancer sequences such as gal 1 and pEFT-1 are useful. Vra-4 alsoprovides a suitable enhancer sequence. Sequences useful as functional“origins of replication” include ars1 and 2 μ circular plasmid. TABLE 2AMINO ACID SEQUENCE ANALYSIS OF ANTI-CD14 mAbs CDRs MAb CDR1 CDR2 CDR33C10-Heavy SYAMS SISSGGTTYYPDNVKG GYYDYHY (SEQ ID NO:10) (SEQ ID NO:11)(SEQ ID NO:12) 28C5-Heavy SDSAWN YISYSGSTSYNPSLKS GLRFAY (SEQ ID NO:13)(SEQ ID NO:14) (SEQ ID NO:15) % homology 17% 38% 14% 3C10-LightRASESVDSFGNSFMH RAANLES QQSYEDPWT (SEQ ID NO:16) (SEQ ID NO:17) (SEQ IDNO:18) 28C5-Light RASESVDSYVNSFLH RASNLQS QQSNEDPYT (SEQ ID NO:19) (SEQID NO:20) (SEQ ID NO:21) 23G4-Light RASESVDSYGKSFMH VASKLES QQNNEDPYT(SEQ ID NO:22) (SEQ ID NO:23) (SEQ ID NO:24) % homology 80% 71% 67%

EXAMPLE 4 In Vivo Treatment with CD14 Antibodies

The pretreatment of rabbits with IFN-γ for three days followed by aninjection of LPS produces a sepsis state in rabbits (G. J. Jurkovich, etal., J. Surg. Res., 51:197-203, 1991). A similar protocol was followedin the in vivo experiments described herein. 5 μg/kg of IFN-γ (specificactivity: 2.5×10⁸ units/mg; 5 μg=1.25×10⁶ units/kg) was injected dailyfor three consecutive days, then following establishment of baselinecardiac output and systemic pressure, an 8 hour infusion of LPS (3 mg/kgtotal dose or 375 μg/kg/hour×8 hours) was started. Subcutaneousinjections of IFN-γ were given 3 days and on the 3rd day baseline datawas collected for at least one hour prior to starting the infusion ofLPS. These animals were maintained on ketamine throughout theexperiment. The rabbit experiments suggested that these animals willbecome somnolent following the LPS infusion. The same establishedprotocol was also utilized in two groups of monkeys. Animals wererandomly assigned to either isotype matched control monoclonalantibodies (MAbs) or a CD14 blocking MAb with the person responsible fortheir care unaware of the treatment protocol. A dose of 5 mg/kg/mAb(isotype or CD14-specific) was given by bolus injection 30′ prior to thestart of the LPS infusion.

Animals were anesthetized with ketamine then arterial and venouscatheters placed in the femoral artery and vein, respectively. Thearterial catheter has a thermistor at its tip for determination ofthermal dilution cardiac output A second lumen on this catheter was usedfor arterial pressure measurement. The venous catheter was used toinfuse drugs, maintenance fluids and for cold injection in the cardiacoutput measurements. Lactated Ringers (3 ml) was used for each cardiacoutput determination.

Blood pressure and cardiac output was recorded every 15 minutesthroughout the baseline period and then every half hour for theremainder of the experiment. Blood was drawn (3 ml) every hour fordetermination of arterial PO₂/PCO₂, pII and protein. This same bloodsample was used for determination of systemic white blood cell countsand differential counts. These animals were resuscitated with lactatedRingers' solution following the infusion of LPS. All animals were givenan infusion of 4 mls/kg as a maintenance infusion and this was increasedas necessary to maintain cardiac output to within 10% of baseline.

A total of six animals each were pretreated with either the IgG1 isotypecontrol or 28C5, and five animals were pretreated with 18E12. All testanimals were challenged with LPS 30 minutes after the infusion ofantibody Seventy-two hours prior to LPS infusion, monkeys were given 3subcutaneous injections of human recombinant interferon gamma (125,000U/Kg) at 24 hour intervals. To measure MAP levels in anesthetizedanimals, arterial and venous catheters were placed in the femoral arteryand vein, respectively as described above.

Mean Arterial Pressure (MAP) of Monkeys Challenged with LPS

The mean arterial pressure (MAP) results reveal that pretreatment with28C5 prevented a significant drop in blood pressure, particularly at the2 hour time point, common in the control group (FIG. 17). However,animals pretreated with 18E12 exhibited this drop in blood pressure at 2hours, yet were able to recover to percents noted in the 28C5 pretreatedanimals. The function of 18E12 differs from that of 28C5 in that onlysignaling events are prevented, not inhibition of LPS binding; a keyfeature of 28C5 as well as 23G4. This difference in function may reflectthe difference noted in the MAP response. Protection by 18E12 mayinvolve late LPS-induced effects. This MAP profile by 18E12 suggeststhat even in the event of physiological responsiveness to LPS (presenceof hypotension) this anti-CD14 mAb is capable of preventing thedeleterious effects noted in the isotype control-treated animals.

Effect of IFN On CD14 Concentration

The pretreatment of animals with interferon-gamma for three consecutivedays, 24 hours apart, had relatively little effect on the CD14concentration (FIG. 18). However, the circulating levels oflipopolysaccharide binding protein (LBP) increased significantly (LBPwas measured by ELISA in which two non-competing monoclonal antibodieswere used to capture and probe test samples); to levels noted duringgram-negative sepsis. It is proposed that the interferon-gamma inducesan acute phase response and sensitizes the animals to doses of LPS whichotherwise would not induce any physiological and/or biochemical changesin these animals.

Lavage/Plasma Ratio of BSA

The Lavage/Plasma ratio of BSA is an indicator of lung damage andaccesses the amount of BSA (injected one hour prior to the terminationof the experiment) that permeates the lung tissue. The lung is one ofthe primary organs affected during endotoxemia. BSA levels weredetermined by an immunoassay utilizing a BSA specific monoclonalantibody. BSA monoclonal antibodies are widely available. In thisinstance, the animals pretreated with 28C5 were protected significantlyfrom the lung damage that was evident in the control-treated group (FIG.19). While 18E12-treated animals were not fully protected from the LPSeffects, as a group they did better than the control animals.

Antibody Half-Life

A question arises as to the antibody half-life, or fate of the antibodydue to its binding to both the soluble circulating form of CD14 and themembrane-associated CD14 present on monocytes and neutrophils. Whencompared to an isotype control antibody which does not recognize humanantigens, the kinetics of clearance are similar for all three groups(FIG. 20).

CD14 Levels in Monkeys Treated with Antibody

CD14 was measured by ELISA in which two non-competing monoclonalantibodies are used to capture and probe test samples. The CD14 levelsin monkeys treated with antibody only (no LPS challenge) weresignificantly higher in the 28C5 versus the control-treated animals(FIG. 21). The reason for this rise is unknown although in vitro studiesdemonstrated that exposure of CD14-bearing cells to anti-CD14 mAb 28C5resulted in higher sCD14 levels; perhaps the antibody enhances theshedding mechanism of this GPI-linked protein. The 18E12-treated animalsshowed an increase at 36 hours which began to drop at 60 hours. Whenantibody-treated animals are challenged with LPS, there is no additionalrise in CD14 levels in the 28C5 group, over what is noted in theantibody-only animal, suggesting that this is clearly andantibody-induced effect.

LBP Levels

The LBP levels rise after interferon-gamma treatment, as was mentionedabove. In the anti-CD14-treated, LPS challenged animals there is aslight lowering of detectable LBP levels versus control (FIG. 22). Thereason for this is not known although it may represent clearance of thecomplexes if the targets are unavailable for binding or transfer of theLPS.

ALT/GPT Levels

Enzyme transaminases ALT/GPT (considered the same enzyme) are indicatorsof liver function and as such were measured to determine if there wasevidence of necrosis. In patients with septic shock, the onset ofhepatic failure is an early event in the MSOF syndrome. Maximum levelsin humans, depending upon the extent of damage, can reach 4000 U/ml.ALT/GPT levels were measured by following the manufacturer'srecommendations in a test kit from Sigma Diagnostics.

While none of the levels recorded here are in the range noted in anextreme human situation, there is a trend in the control group forelevated enzyme levels during the 24 hour time course of the experiment.Normal levels for ALT/GPT in monkeys treated with antibody only, rangedfrom a mean of 18 U/ml (T=0) to 52.2 U/ml (T=24), with the elevation atT=24 attributed to the anesthesia used throughout the study (ketamine).The two anti-CD14 treated groups followed a similar course; a mean of16.5 U/ml at T=0 to 64.6 U/ml at T=24. The mean of the control group was22.8 U/ml at T=0 to 98 U/ml at T=24. A twenty-four hour time course maynot allow one to determine if the elevation in the control group ofanimals would continue (FIG. 23).

Soluble E-Selectin Levels

Soluble E-selectin levels were measured by Parameter ELISA kit (BritishBio-technology Products, Ltd.) to determine if blocking the CD14receptor would somehow prohibit release of soluble E-selectin fromendothelial cells. E-selectin expression on the surface of endothelialcells is an indicator of activation of these cells and occurs as aconsequence of TNF, IL-1 or LPS stimulation. Soluble E-selectin levelswere elevated to similar levels, at 24 hours, in all groups (FIG. 24).

IL-1, IL-6, IL-8 and TNF Levels

The cytokine response to LPS challenge was evaluated using Quantikine™kits (R&D Systems), performed according to the manufacturer'sspecifications, in all groups of animals. The assay is an immunoassaywith a solid phase ELISA format. The TNFα assay was a Biokine® enzymeimmunoassay kit (T Cell Diagnostics) and was performed according to themanufacturer's specifications. It is known that TNFα and IL-1β are keymediators of the inflammatory response induced as a consequence of LPSstimulation. In the anti-CD14 treated groups, the TNFα and IL-1βresponses were reduced versus the control treated group with 18E12exhibiting the lowest level of expression of these inflammatorycytokines. Also, the peak TNF response was delayed by an hour in bothanti-CD14 groups, the significance of this finding is no known at thistime (FIG. 25).

It is known that IL-1, IL-6 and IL-8 peak later than TNF in humansepticemia, which is in line with the observations that release of IL-1,IL-6 and IL-8 is largely dependent on TNF generation. One cytokine whichhas been correlated with mortality in humans diagnosed withgram-negative sepsis is elevated IL-6 levels. IL-6 coordinates variousaspects of the host defense against tissue injury. In the present model,28C5 exhibited the lowest level of IL-6 in response to LPS. Animalspretreated with 18E12 also had lower levels than the control group, yetnot as low as 28C5. The IL-8 response, while not significantly lower inthe anti-CD14 groups, was reduced slightly (FIG. 26). IL-8 levels inbaboon models were shown to be correlated with TNF levels; reduced TNFresulted in reduced IL-8 levels. IL-8 has chemoattractant andgranulocyte activation properties. Relatively preserving the native IL-8response, as noted in the anti-CD14 treated groups, may keep intactthese important mediators of the host response to LPS.

SUMMARY OF SEQUENCES

SEQ ID NO:1 is the nucleic acid and deduced amino acid sequence of the28C5 heavy chain.

SEQ ID NO:2 is the deduced amino acid sequence of the 28C5 heavy chain.

SEQ ID NO:3 is the nucleic acid and deduced amino acid sequence of the28C5 light chain.

SEQ ID NO:4 is the deduced amino acid sequence of the 28C5 light chain.

SEQ ID NO:5 is the nucleic acid and deduced amino acid sequence of the18E12 heavy chain.

SEQ ID NO:6 is the deduced amino acid sequence of the 18E12 heavy chain.

SEQ ID NO:7 is the nucleic acid and deduced amino acid sequence of the18E12 light chain.

SEQ ID NO:8 is the deduced amino acid sequence of the 18E12 light chain.

SEQ ID NO:9 shows the nucleic acid sequence which encodes the humansoluble CD14 receptor.

SEQ ID NO:10 is the amino acid sequence of CDR1 of 3C10 heavy chain.

SEQ ID NO:11 is the amino acid sequence of CDR2 of 3C10 heavy chain.

SEQ ID NO:12 is the amino acid sequence of CDR3 of 3C10 heavy chain.

SEQ ID NO:13 is the amino acid sequence of CDR1 of 28C5 heavy chain.

SEQ ID NO:14 is the amino acid sequence of CDR2 of 28C5 heavy chain.

SEQ ID NC:15 is the amino acid sequence of CDR3 of 28C5 heavy chain.

SEQ ID NO:16 is the amino acid sequence of CDR1 of 3C10 light chain.

SEQ ID NO:17 is the amino acid sequence of CDR2 of 3C10 light chain.

SEQ ID NO:18 is the amino acid sequence of CDR3 of 3C10 light chain.

SEQ ID NO:19 is the amino acid sequence of CDR1 of 28C5 light chain.

SEQ ID NO:20 is the amino acid sequence of CDR2 of 28C5 light chain.

SEQ ID NO:21 is the amino acid sequence of CDR3 of 28C5 light chain.

SEQ ID NO:22 is the amino acid sequence of CDR1 of 23G4 light chain.

SEQ ID NO:23 is the amino acid sequence of CDR2 of 23G4 light chain.

SEQ ID NO:24 is the amino acid sequence of CDR3 of 23G4 light chain.

SEQ ID NO:25 is the amino acid sequence of the 23G4 light chain.

1. A hybrid cell line which produces a monoclonal antibody, themonoclonal antibody being specifically reactive with cell surface CD14and inhibiting CD14 mediated cell activation by a ligand.